Isoflavone-containing composition

ABSTRACT

This invention provides a composition comprising a daidzein-containing substrate and a strain of micro-organism capable of metabolizing daidzein to equol as essential ingredients. This composition is effective in the prevention and alleviation of unidentified clinical syndrome inclusive of menopausal syndrome in middle-aged to elderly women for which no effective means of prevention or alleviation has heretofore been available.

This is a divisional of application Ser. No. 10/752,674 filed Jan. 8,2004, which is a divisional of application Ser. No. 09/485,320 filedFeb. 8, 2000 (now U.S. Pat. No. 6,716,424), which in turn is a §371National Stage entry of PCT/JP98/03460, filed Aug. 4, 1998. The entiredisclosure of the prior applications, application Ser. Nos. 10/752,674and 09/485,320 and PCT/JP98/03460, is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to an isoflavone-containing compositionand more particularly to a novel composition either comprising adaidzein-containing substance and a strain of microorganism capable ofmetabolizing daidzein to equol or comprising equol obtained by causingsaid strain of microorganism to act upon said daidzein-containingsubstance, which composition is useful for the prevention andalleviation of unidentified clinical syndrome and conditions associatedwith the menopause in middle-aged to elderly women.

BACKGROUND ART

The documented collaborative research of National Cancer Center of Japanand Helsinki University (Finland) attributes the low incidence ofgender-specific neoplastic diseases such as carcinoma of the prostate inmen and carcinoma of the breast or ovary in women among the Japanese ascompared with the European and American people to the greater intake bythe Japanese of soybean-derived foods containing various isoflavonoidsand the consequent well-coordinated balance of hormones (H. Adlercreutz,et al., (1992) Lancet, 3, 1233; H. Adlercreutz, et al., (1992) Lancet,342, 1209-1210).

Recently, there has been a mounting interest in the fact thatisoflavonoids have estrogen (female hormone)-like activity (A. Molteni,et al., (1995) J. Nutr., 125, 751S-756S), and it has been reported thatthese compounds are effective in osteoporosis which develops after themenopause when estrogen secretions have subsided or ceased (D. Agnusdei,et al., (1995) Bone and Mineral, 19 (Supple), S43-S48) as well as inmenopausal syndrome (D. D. Baird, et al., (1995) J. Clin. Endocrinol,Metab., 80, 1685-1690; A. L. Murkies, et al., (1995) Maturitas., 21,195-198).

According to the result of a survey undertaken by Margaret Lock (M.Lock, et al., (1988) Maturitas., 10, 317-332), the incidence ofmenopausal syndrome among the Japanese women is extremely low ascompared with the Canadian counterparts. Based on the report, H.Adlercreutz and coworkers conjecture that the Japanese women ingestlarge amounts of processed soybean foods such as tofu, miso, soy sauce,etc. and, hence, the plant estrogens (isoflavonoids) occurring in thosefoods are responsible for the low incidence of menopausal syndrome.Comparing the urinary excretions (24-hour urine) which are known toreflect the amount of absorption of isoflavonoids actually ingested, thesame authors further report that compared with the Western women, theurinary excretions in the Japanese women are tens of times as high (C.Herman, et al., (1995), J. Nutr., 1, 757S-770S).

It is, thus, considered that the intake of isoflavonoids such asdaidzein, genistein, daidzin, genistin, etc. is effective for thealleviation and prevention of postmenopausal osteoporosis and menopausalsyndrome. Particularly, the postmenopausal life expectancy in women hasreportedly increased to more than 30 years owing to the recent trendtoward longevity and the alleviation and prevention of various diseasesand symptoms which may develop after the menopause have importantmeanings in that they would lead to improvements in quality of life(QOL).

However, the above report, i.e. the survey report on the amount ofintake of isoflavonoids and the urinary excretions of isoflavonoids inthe middle-aged to elderly women in Japan reflects the results generatedin a limited rural area and no substantive information is available.Moreover, the correlation between the frequency of menopausal syndromeand the amount of intake of isoflavonoids has not been squarely analyzedand revealed.

Therefore, the object of the present invention is to provide a novelcomposition which is effective for the prevention and alleviation of theso-called unidentified clinical syndrome in middle-aged to elderlywomen, inclusive of menopausal syndrome, for which no effective means ofprevention or alleviation has been available.

To accomplish the above object, the inventors first conducted a dietarysurvey, determination of urinary excretions of isoflavonoids, and aquestionnaire survey about menopausal syndrome (unidentified clinicalsyndrome) in perimenopausal women in a broad geographical area includingurban communities.

According to the results of the above investigation conducted in 116women aged between 40 and 60 who belonged to Fukuoka DietitianAssociation, the average amounts of intake of isoflavonoids were 9mg/day for daidzein and 13 mg/day for genistein. The average urinaryexcretions of isoflavonoids were 19.6 μmol/day for daidzein and 10.0μmol/day for genistein, and the average excretions of equol, ametabolite of daidzein, was 11.9 μmol/day (mean of subjects in whom itwas detected). Incidentally, although daidzein and genistein weredetected in all the subjects, equol was detected only in 46 (51.6%) ofthe 95 subjects.

Furthermore, women with paramenia and those within 5 years of themenopause being taken together as menopausal subjects, a questionnairesurvey was conducted using 17 items which are in routine use in thediagnosis of menopausal syndrome [17 items as a modification ofKupperman menopausal index (Kupperman H. S., et al., (1953), J. Clin.Endocrinol. Metabol., 13, 688-703), i.e. 1. hot flushes, 2.perspiration, 3. local sensation of cold, 4. shortness of breath, 5.numbness of limbs, 6. hypesthesia, 7. difficulty in falling asleep, 8.fitful sleep, 9. irritability, 10. nervousness, 11. melancholy, 12.vertigo, nausea, 13. weakness (fatigue), 14. stiff shoulders, pain injoints, muscular pain, 15. headache, 16. palpitation, 17. tinglingsensation] and the simplified menopausal index (SMI) was calculated.With subjects with SMI values not less than 20 being taken as a group ofhigh climacteric symptoms and those with SMI values not greater than 19as a group of low climacteric symptoms, the amount of intake ofisoflavonoids and the urinary excretion of isoflavonoids wererespectively compared between the groups.

As a result, whereas no intergroup difference was found in the amount ofintake of daidzein, the amount of intake of genistein tended to be lowerin the group of high climacteric symptoms at p=0.0643. With regard tothe urinary excretions of isoflavonoids, no intergroup difference wasfound whether for daidzein or for genistein but the excretions of equalwere significantly low (p<0.01) in the group of high climactericsymptoms.

From the above results, the inventors found that unidentified clinicalsymptoms in menopausal women are more closely related to the amount ofintake of genistein and the urinary excretion of equal, among variousisoflavonoids.

In the past the relationship of the amounts of intake and urinaryexcretion of isoflavonoids as a whole to their physiological effect hasbeen discussed without regard to specific kinds of isoflavonoids such asdaidzein and genistein but the results of the survey conducted by theinventors in the Japanese middle-aged and elderly women made it clearthat not only the amounts of intake and urinary excretion ofisoflavonoids in general but also the amount of intake of genistein andthe urinary excretion of equol, particularly the rate of metabolicconversion of daidzein to equol, are closely related to unidentifiedclinical climacteric symptoms in menopausal women.

In another study undertaken by the inventors in healthy adult volunteers(25-33 years of age), it was found that the urinary excretions ofisoflavonoids (daidzein and genistein) after single ingestion of soymilk, a representative isoflavonoid-containing food, are increased in adose-related fashion but in subjects who showed no urinary excretion ofequol, equol was not detected in the urine even when the amount ofintake of soy milk was increased two-fold, indicating the existence ofindividual difference in the metabolic pathway from daidzein to equol.

It is known that equol, which is a metabolite of daidzein, is notdetected in isoflavonoid-containing foods such as processed soybeanproducts nor is it taken into the body from foods in ordinary diets (K.Reinli, et al., (1996), Nutr. Cancer, 26, 123-148).

Based on the above findings the inventors did further research and, as aresult, succeeded in the development of a novel composition whichcomprises a strain of microorganism having the ability (metabolicactivity) to elaborate equol from daidzein and either daidzein or asuitable substance containing daidzein in combination, and a novelcomposition which comprises equol obtained by causing said strain ofmicroorganism to assimilate daidzein. The inventors then discovered thatthe intake of whichever of the above compositions is effective in theprevention and alleviation of unidentified clinical syndrome inmiddle-aged and older women and have accordingly developed the instantinvention.

DISCLOSURE OF INVENTION

The present invention in a first aspect provides a composition, in theform of a food or a pharmaceutical product, which comprises adaidzein-containing substance and a strain of microorganism capable ofmetabolizing daidzein to equol as essential ingredients (whichcomposition will hereinafter be referred to as “isoflavone-containingcomposition”).

The present invention in a second aspect provides a composition, in theform of a food or a pharmaceutical product, which comprises equol whichis obtained by causing a strain of microorganism capable of metabolizingdaidzein to equol to act upon a daidzein-containing substance (whichcomposition will hereinafter be referred to as “equol-containingcomposition”).

The present invention further provides said isoflavone-containingcomposition and equol-containing composition wherein the strain ofmicroorganism capable of metabolizing daidzein to equol is at least onemember selected from the group consisting of Bacteroides ovatus,Streptococcus intermedius and Streptococcus constellatus and moreparticularly said isoflavone-containing composition and equol-containingcomposition wherein said strain of microorganism is at least one memberselected from the group consisting of Bacteroides E-23-15, which hasbeen deposited as FERM BP-6435, Streptococcus E-23-17, which has beendeposited as FERM BP-6436, and Streptococcus A6G-225, which has beendeposited as FERM BP-6437.

The present invention further provides said isoflavone-containingcomposition and equol-containing composition which further contain atleast one ingredient that favors the maintenance and growth of saidstrain of microorganism, for example at least one substance selectedfrom the group consisting of galactosylsucrose, soybean-oligosaccharide,lactulose, lactitol and fructo-oligosaccharide.

The present invention further provides said isoflavone-containingcomposition and equol-containing composition wherein saiddaidzein-containing substance further contains at least one memberselected from the group consisting of genistein, daidzin and genistin,more preferably soya isoflavone.

The present invention further provides said isoflavone-containingcomposition and equol-containing composition for the prevention andtreatment of unidentified clinical syndrome in middle-aged to elderlywomen, inclusive of menopausal syndrome.

The present invention further provides said isoflavone-containingcomposition and equol-containing composition in the form of a food whichis selected from the group consisting of drinks, dairy products,fermented milk, bars, granules, powders, capsules and tablets.

The present invention further provides said isoflavone-containingcomposition and equol-containing composition in the form of apharmaceutical product which is selected from the group consisting ofaqueous solutions, emulsions, granules, powders, capsules and tablets.

The present invention in a further aspect provides a method forprevention and treatment of unidentified clinical syndrome or menopausalsyndrome in middle-aged to elderly women which comprises administeringan effective amount of said isoflavone-containing composition orequol-containing composition to a middle-aged or elderly woman in whomsaid prevention or treatment are needed.

The present invention further provides the use of a microorganismcapable of utilizing a daidzein-containing substrate or daidzein toproduce equol for the production of said isoflavone-containingcomposition and equol-containing composition which are effective for theprevention and treatment of unidentified clinical syndrome or menopausalsyndrome in middle-aged to elderly women.

The present invention further provides a method of producing equol whichcomprises causing a strain of microorganism capable of metabolizingdaidzein to equol to act upon daidzein.

The present invention further provides a strain of microorganismselected from the group consisting of Bacteroides E-23-15, which hasbeen deposited as FERM BP-6435, Streptococcus E-23-17, which has beendeposited as FERM BP-6436, and Streptococcus A6G-225, which has beendeposited as FERM BP-6437.

The isoflavone-containing composition of the invention is now describedin detail.

In the isoflavone-containing composition of the invention, adaidzein-containing substance is used as one of its essentialingredients. This daidzein-containing substance includes not onlydaidzein as such but also daidzin which is a glycoside of daidzein and avariety of substances containing daidzein and/or daidzin. The daidzeinpresent itself chiefly in soybean, kudzu and the like raw foods, theirprocessed products such as tofu, aburage, soy milk, etc. and theirfermentation products such as natto, soy sauce, miso, tempeh, etc. Inthe present invention, any of such raw foods, processed products andfermentation products can be used as said daidzein-containing substance.Particularly, the substances contain not only daidzein but also otherisoflavonoids having estrogen-like activity, such as genistein, daidzin,genistin, etc., biochain A and formonetin which is a partiallymethylated precusor of genistein and daidzein, respectively, etc. andcan be used with advantage for the purpose of the invention.

The daidzein-containing substance which is preferred for the practice ofthe present invention further includes soya isoflavone derived fromsoybeans, for example commercial products such as “Fujiflavone (tradename) P10” from Fujicco, and isoflavonoids derived from plants such asred clove, alphalpha, etc.

In the isoflavone-containing composition of the invention, a strain ofmicroorganism having an ability (metabolic activity) to produce equolfrom daidzein is used as the other essential ingredient. Themicroorganism includes those belonging to Bacteroides ovatus,Streptococcus intermedius, and Streptococcus constellatus. Particularlypreferred among such microorganisms are Bacteroides E-23-15 (FERMBP-6435), Streptococcus E-23-17 (FERM BP-6436) and Streptococcus A6G-225(FERM BP-6437), all of which were isolated from human stools anddeposited for accession by the inventors.

The bacteriological characteristics of those strains of microorganismsare now described in detail.

(1) Bacteroides E-23-15, FERM BP-6435 I. Cultural Characters

When cultured anaerobically using an anaerobic jar stuffed with steelwool at 37° C. for 48 hours, this strain gives good to moderate growthon Eggerth-Gagnon (EG) agar, Blood Liver (BL) agar, or Gifu AnaerobicMedium (GAM). The colonies are circular, protuberant in a convex manner,with both the surface and peripheral edges being glabrous to slightlycoarse. The colony color is grayish white on EG agar or grayish brown onBL agar. Morphologically it is a gram-negative rod and showspolymorphism ranging from coccobacillus, single rod, elongated rod,etc., but the cells occur singly and not in chains. No sporogenesis isfound.

II. Physiological Characteristics

-   -   (1) Optimum temperature for growth: 37° C.    -   (2) Optimum pH for growth: 7.0    -   (3) Liquefaction of gelatin: +    -   (4) Hydrolysis of soluble starch: +    -   (5) Hydrolysis of esculin: +    -   (6) Indole production: −    -   (7) Urease: −    -   (8) Catalase: −    -   (9) Assimilation of carbon sources:        -   L-arabinose +        -   D-xylose +        -   D-glucose +        -   Sucrose +        -   L-Rhamnose +        -   D-Raffinose +        -   D-Mannitol +        -   Indole +        -   Lactose +        -   Maltose +        -   Salicin +        -   Gelatin +        -   Glycerin +        -   D-cellobiose +        -   D-mannose +        -   D-melezitose +        -   D-sorbitol +        -   D-trehalose +    -   (10) Organic acid composition after utilization of peptone or        glucose:

Using PYF (peptone-yeast extract-Fildes) medium (containing about 5% ofpeptone), which is utilized in sugar fermentation test, and PYF mediumsupplemented with 0.5% final concentration of glucose, the strain wascultured anaerobically at 37° C. for 72 hours and the organic acids inthe resulting culture were assayed by HPLC. The results (unit: mM) areshown below.

Glucose-PYF Organic acid PYF culture Culture Maleic acid 0.02 1.19Succinic acid 0.01 3.20 Lactic acid 0.01 4.94 Formic acid 0.03 0.66Acetic acid 0.29 2.62 Pyroglutamic acid 0.01 nd Propionic acid nd ndi-Butyric acid 1.71 0.23 n-Butyric acid 0.36 nd i-Valeric acid nd 0.19n-Valeric acid nd nd nd = not detected

The above morphological and biochemical characteristics, sugarfermentation test and organic acid production spectrum suggested thatthis strain was either of the gram-negative rods Bacteroides ovatus andBacteroides uniformis but was decided to be a microorganism belonging toBacteroides ovatus in view of its ability to utilize rhamnose.Accordingly this strain was named Bacteroides E-23-15 and deposited withNational Institute of Bioscience and Human Technology (NIBH, Higashi1-1-3, Tsukuba-shi, Ibaraki, Japan) as of Jul. 7, 1997 under theaccession number of FERM P-16312. This deposit was converted to aBudapest deposit on Jul. 22, 1998 and assigned the accession number ofFERM BP-6435.

(2) Streptococcus E-23-17 (FERM BP-6436) I. Cultural Characteristics

When cultured anaerobically in an anaerobic jar stuffed with steel woolat 37° C. for 48 hours, this strain gives good to moderate growth on EGagar, BL agar or GAM. The colonies are circular and conical toprotuberant in a centrally convex fashion, and have a ground glass-liketo granular texture with a smooth or slightly coarse edge. The colonieson EG agar are transparent to grayish brown. Morphologically it is agram-positive coccus, ellipsoidal or with slightly pointed ends. Thecells occur singly or are diplococcal, forming irregular masses. Nochain is formed. The strain is not sporogenic.

II. Physiological Characteristics

-   -   (1) Optimum temperature for growth: 37° C.    -   (2) Optimum pH for growth: 7.0    -   (3) Liquefaction of gelatin: −    -   (4) Hydrolysis of soluble starch: −    -   (5) Hydrolysis of esculin: +    -   (6) Indole production: −    -   (7) Urease: −    -   (8) Catalase: −    -   (9) Assimilation of carbon sources:        -   L-arabinose +        -   D-xylose −        -   D-glucose +        -   Sucrose −        -   L-Rhamnose +        -   D-Raffinose −        -   D-Mannitol +        -   Indole −        -   Lactose +        -   Maltose +        -   Salicin +        -   Gelatin −        -   Glycerin −        -   D-cellobiose +        -   D-mannose +        -   D-melezitose −        -   D-sorbitol +        -   D-trehalose +    -   (10) Organic acid composition after utilization of peptone or        glucose:

Using PYF (peptone-yeast extract-Fildes) medium (containing about 5% ofpeptone), which is utilized in sugar fermentation test, and PYF mediumsupplemented with 0.5% final concentration of glucose, the strain wascultured anaerobically at 37° C. for 72 hours and the organic acids inthe resulting culture were assayed by HPLC. The results (unit: mM) areshown below.

Glucose-PYF Organic acid PYF culture Culture Maleic acid 0.04 NdSuccinic acid 2.37 0.02 Lactic acid 0.02 nd Formic acid 0.03 0.03 Aceticacid 3.32 0.07 Pyroglutamic acid 0.03 nd Propionic acid 3.24 ndi-Butyric acid 4.17 1.11 n-Butyric acid nd nd i-Valeric acid 4.50 ndn-Valeric acid nd nd nd = not detected

The above morphological and biochemical characteristics, sugarfermentation test and organic acid production spectrum suggest that thisstrain belongs to either of the gram-positive cocci Luminococcusproductus and Streptococcus constellatus but the strain differentiatesitself from the type culture strain of Luminococcus productus in theability to utilize sucrose, D-xylose and D-raffinose. Therefore, theinventors named the strain Streptococcus E-23-17 and deposited it withNational Institute of Bioscience and Human Technology (NIBH, Higashi1-1-3, Tsukuba-shi, Ibaraki, Japan) as of Jul. 7, 1997 under theaccession number of FERM P-16313. This deposit was subsequentlyconverted to a Budapest deposit as of Jul. 22, 1998 and assigned withthe accession number of FERM BP-6436.

(3) Streptococcus A6G-225 (FERM BP-6437) I. Cultural Characteristics

When cultured anaerobically using an anaerobic jar stuffed with steelwool at 37° C. for 48 hours, this strain shows good to moderate growthon EG agar, BL agar or GAM. The colonies are circular, conical toprotuberant in a centrally convex fashion and have ground glass-like togranular texture with a smooth or slightly coarse peripheral edge. Thecolonies on EG agar are transparent to grayish white. Morphologically itis a gram-positive coccus, ellipsoidal or with slightly pointed ends.The cells occur singly or are diplococcal, forming irregular masses. Nochain is formed. No sporogenesis is found, either.

II. Physiological Characteristics

-   -   (1) Optimum temperature for growth: 37° C.    -   (2) Optimum pH for growth: 7.0    -   (3) Liquefaction of gelatin: −    -   (4) Hydrolysis of soluble starch: −    -   (5) Hydrolysis of esculin: +    -   (6) Indole production: −    -   (7) Urease: −    -   (8) Catalase: −    -   (9) Assimilation of carbon sources:        -   L-arabinose −        -   D-xylose −        -   D-glucose +        -   Sucrose +        -   L-Rhamnose −        -   D-Raffinose +        -   D-Mannitol −        -   Indole −        -   Lactose +        -   Maltose +        -   Salicin +        -   Gelatin −        -   Glycerin −        -   D-cellobiose +        -   D-mannose +        -   D-melezitose −        -   D-sorbitol −        -   D-trehalose −    -   (10) Organic acid composition after utilization of peptone or        glucose:

Using PYF (peptone-yeast extract-Fildes) medium (containing about 5% ofpeptone), which is utilized in sugar fermentation test, and PYF mediumsupplemented with 0.5% final concentration of glucose, the strain wascultured anaerobically at 37° C. for 72 hours and the organic acids inthe resulting culture were assayed by HPLC. The results (unit: mM) areshown below.

Glucose-PYF Organic acid PYF culture culture Maleic acid nd Nd Succinicacid 0.21 0.03 Lactic acid nd 35.36 Formic acid 0.55 1.66 Acetic acid1.35 0.54 Pyroglutamic acid nd nd Propionic acid nd nd i-Butyric acid2.04 nd n-Butyric acid nd nd i-Valeric acid nd nd n-Valeric acid nd ndnd = not detected

The above morphological and biochemical characteristics, sugarfermentation test and organic acid production spectrum suggest that thisstrain belongs to the gram-positive Streptococcus intermedius but thestrain differentiates itself from the type culture strain of S.intermedius in the ability to utilize L-rhamnose and D-trehalose.Therefore, the inventors named the strain Streptococcus A6G-225 anddeposited it with National Institute of Bioscience and Human Technology(NIBH, Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) as of Jul. 7, 1997,with the accession number of FERM P-16314 assigned. This deposit wassubsequently converted to a Budapest deposit as of Jul. 22, 1998 andassigned with the accession number of FERM BP-6437.

The above three strains of microorganisms isolated by the inventors havethe ability to utilize daidzein to elaborate equol as their mostoutstanding characteristic. The daidzein includes daidzein as theaglycone of an isoflavone glycoside such as daidzin. Daidzin is utilizedby said microorganisms to give daidzein, and equol is then produced fromthis daidzein.

There has been no report on such a microorganism capable of producingequol. Therefore, the present invention further provides novel strainsof microorganisms having the ability to produce equol.

The above strain of microorganism for use as an essential ingredient ofthe isoflavone-containing composition of the invention may generally bethe live microorganism as such. However, it is not limited thereto butincludes its culture, a crude or purified product thereof, and theirlyophilyzates. Its proportion is not particularly restricted but can bejudiciously selected according to the kind of microorganism, among otherfactors. For example, in the case of Streptococcus intermedius infermented milk, the bacterial count is preferably controlled within therange of about 10⁸ to 10⁹ cells/ml. The bacterial count is determined byinoculating an agar medium with a diluted sample, incubating theinoculated medium anaerobically at 37° C. and counting the coloniesformed. In the case of other strains of microorganisms, too, the countdetermined in the above manner can be used as a rule of thumb.

The isoflavone-containing composition of the invention furtherpreferably contains a nutrient component particularly suited to themaintenance and growth of the particular strain of microorganism. Thenutrient component includes various oligosaccharides such asgalactosylsucrose, soybean-oligosaccharide, lactulose, lactitol,fructo-oligosaccharide, and galacto-oligo-saccharide. The formulatingamount of such nutrients is not particularly restricted but generally ispreferably selected from the range of about 1 to 3 weight % based on thetotal composition of the invention.

The composition of the invention is generally prepared by blendingpredetermined amounts of said essential ingredients and other optionalingredients and processing the mixture into a suitable food form orpharmaceutical dosage form, such as drinks, dairy products, fermentedmilk, bars, granules, powders, capsules, tablets, etc. for food use oraqueous solutions, emulsions, granules, powders, capsules, tablets, etc.for pharmaceutical use. The production of such dosage forms can becarried out in the conventional manner. The carrier for use in themanufacture of such dosage forms includes edible carriers andpharmaceutically acceptable excipients and diluents. Particularly in thecase of a food form, a palatable and taste-improving carrier ispreferred.

The particularly preferable examples of carrier include such maskingagents as trehalose (manufactured by Hayashibara), cyclodextrin,Benekote BMI (manufactured by Kao Corporation), etc.

The blending ratio of said daidzein-containing substance, specificstrain of microorganism and optional ingredients used for themaintenance and growth of the microorganism is not particularlycritical. However, based on 100 g of the composition of the invention,the proportion of the daidzein-containing substance is preferably withinthe range of about 10-50 mg of daidzein contained therein. On the samebasis, the proportion of the microorganism is preferably 10⁹ to 10¹⁰cells (as viable cells) and that of the oligosaccharide is preferablywithin the range of about 1-5 g.

Since the isoflavone-containing composition of the invention contains astrain of microorganism (primarily live cells) as mentioned above, thecomposition preferably should not be subjected to heating and/orpressurization in the course of processing into final products.Therefore, in processing the composition of the invention into suchdosage forms as bars, granules, powders, tablets, etc., it is preferableto add the microorganism as lyophilized cells as such or lyophilizedcells coated with a suitable coating agent.

The composition of the invention may be optionally supplemented withvarious other food ingredients having nutritional values or variousadditives which are conventionally used in the manufacture ofpharmaceutical products. The food ingredients mentioned above includecalcium, vitamin B, vitamin D, vitamin C, vitamin E and vitamin K(particularly MK-7 (menaquinone-7) derived from Bacillus natto). Otherexamples of the substances that can be added include zinc and selenium.

The resulting isoflavone-containing composition of the invention isuseful for the prevention and treatment of unidentified clinicalsyndrome, postmenopausal osteoporosis and other menopausal syndrome andsymptoms in middle-aged and older women. Such prevention and treatmentare achieved by administering or ingesting an effective amount of theabove composition of the invention to a middle-aged or elderly woman whoneeds such prevention or treatment. The effective amount of thecomposition is not particularly restricted insofar as the prevention andtreatment of unidentified clinical syndrome, postmenopausal osteoporosisor menopausal syndrome can be achieved with it. In general, theeffective amount is preferably such that about 10-50 mg/day of daidzeinand at least about 10 mg/day of genistein can be taken.

The equol-containing composition of the invention is now described indetail.

The equol-containing composition of the invention comprises equolobtainable by causing a strain of microorganism capable of utilizingdaidzein to produce equol to act upon a daidzein-containing substance.

The strain of microorganism may be the same as that of theabove-described isoflavone-containing composition of the invention. Thedaidzein-containing substance on which said microorganism is caused toact can also be the same as that mentioned for the isoflavone-containingcomposition of the invention, thus including an isolated and purifiedform of daidzein, food materials containing it, processed matters orfermentation products thereof, soya isoflavone and isoflavones derivedfrom kudzu, red clove, alphalpha, etc., products containing suchisoflavones, for example tofu, soy milk, boiled soybeans, natto, soybeanhypocotyl extract, etc.

The equol-containing composition of the invention is very safe becausethe active ingredient thereof is a native substance as mentioned above.Moreover, since it is prepared by using a microorganism, the compositionis not only free from contamination with chemicals and the likecontaminants but also advantageous in that it can be obtained in highyield and at low production cost.

The equol-containing composition of the invention can be produced byconventional fermentation technology utilizing said daidzein-containingsubstance, preferably soya isoflavone or a food material containing it,as the substrate.

More particularly, the technology comprises sterilizing the substrate insolution form, adding the predetermined strain of microorganism thereto,and incubating the mixture at 37° C. either under anaerobic conditionsor under aerobic stationary conditions for about 48-96 hours to letfermentation proceed. (Where necessary, a pH control agent, a reducingsubstance (e.g. yeast extract, vitamin K₁) can be added).

Taking Streptococcus intermedius as an example, the above culturalprocess can be more preferably carried out as follows. First, daidzeinis dissolved in the range of 0.01-0.5 mg/ml in Modified GAM (ModifiedGifu Anaerobic Medium) for culture of anaerobic bacteria. A seed cultureprepared by growing the microorganism in Modified GAM for about 14 hoursis then inoculated into the above daidzein-containing Modified GAM. Theinoculum size may be 1/100 by volume of the medium. The incubated mediumis incubated aerobically at 37° C. under stationary conditions for 48-96hours.

The present invention further provides a method of producing equolutilizing such a strain of microorganism.

In the above fermentation system, there may be incorporated a nutrientwhich is particularly suited for the maintenance and growth of themicroorganism. The nutrient includes oligosaccharides such asgalactosyl-sucrose, soybean-oligosaccharide, lactulose, lactitol,fructo-oligosaccharide, and galacto-oligosaccharide. The amount of saidnutrient is not particularly restricted but is preferably selected fromthe range of generally about 1-3 weight % based on the total compositionof the invention.

The desired equol-containing culture broth can thus be obtained.

Isolation and purification of equol from the fermentation broth can becarried out in the conventional manner. A typical procedure may compriseadsorbing the fermentation broth on an ion exchange resin (e.g. DIAIONHP20, Mitsubishi Kasei Corporation), eluting the objective substancewith methanol, and concentrating the active fraction to dryness toprovide crude equol.

The equol-containing composition of the invention can be produced, in asuitable food form or pharmaceutical dosage form, by formulating theequol-containing culture broth prepared as above or equol isolatedtherefrom with other optional food materials.

The food form includes drinks, milk products, fermented milk, bars,granules, powders, capsules, and tablets. The pharmaceutical dosage formincludes aqueous solutions, emulsions, granules, powders, capsules andtablets. Those food or pharmaceutical dosage forms can each bemanufactured by the established technology. The carrier for use in themanufacture of such forms may be any of edible carriers andpharmaceutically acceptable excipients and diluents. Particularly in thecase of foods, the carrier is preferably a palatable, taste-improvingcarrier.

The amount of equol in the resulting composition of the invention is notparticularly restricted but can be determined according to the intendedfood form or pharmaceutical dosage form. Usually, however, based on 100g of the total composition, the amount of equol is preferably about10-50 mg.

The amount of intake of the composition of the invention is notparticularly restricted but can be generally selected so that theurinary excretions of equol after ingestion of the composition will notbe less than 5 μmole/day.

The equol-containing composition of the invention is useful for theprevention and treatment of unidentified clinical syndrome inmiddle-aged to elderly women, typically symptoms of postmenopausalosteoporosis and menopausal syndrome.

BEST MODE FOR CARRYING OUT THE INVENTION

For a further detailed description of the invention, examples ofpreparation of the isoflavone-containing composition andequol-containing composition of the invention and an example ofproduction of equol are presented below. It being to be understood,however, that the scope of the invention is not limited by thoseexamples.

EXAMPLE 1 Preparation of a Drink

The ingredients according to the following recipe were weighed andblended to provide the composition of the invention in the form of abeverage.

Fermentation broth of water-soluble soybean protein

Fermentation broth of water-soluble soybean protein 10 mlGalactosylsucrose (55% content) 10.0 g Vitamins & minerals q.s. Flavorq.s. Water q.s. Total 150 ml

The above fermentation broth of water-soluble soybean protein wasprepared by dissolving 2.2 g of water-soluble soybean protein in 10 mlof water; adding 10⁸ cells of Streptococcus A6G-225 (FERM BP-6437)thereto, and incubating the mixture at 37° C. for 48 hours.

EXAMPLE 2 Preparation of a Fermented Milk

The ingredients according to the following recipe were weighed andblended to provide the isoflavone-containing composition of theinvention in the form of fermented milk.

Water-soluble soybean protein 2.2 g Galactosylsucrose (55% content) 10.0g Streptococcus A6G-225-fermented milk 100 ml Vitamins & minerals q.s.Flavor q.s. Water q.s. Total 50 ml

The water-soluble soybean protein contained about 3-4% of daidzein (asanalyzed by high-performance liquid chromatography; the same applieshereinafter). The Streptococcus A6G-225-fermented milk was prepared byadding 10⁸ cells of Streptococcus A6G-225 (FERM BP-6437) to 1 liter ofmilk and incubating the mixture at 37° C. for 24 hours.

EXAMPLE 3 Preparation of a Fermented Soy Milk Lyophilizate

Using 1 ml of a suspension of about 10⁷ cells/ml of StreptococcusA6G-225 (FERM BP-6437), 100 g of soy milk was caused to undergo lacticacid fermentation at 37° C. for 24 hours to provide equol. This productwas lyophilized. The equol content of this freeze-dried powder was0.1-0.3 weight %.

The above powder and other ingredients according to the following recipewere weighed and blended to provide the composition of the invention inthe form of a fermented soy milk lyophilizate.

Fermented soy milk lyophilizate 2.2 g Excipient q.s. Vitamins & mineralsq.s. Flavor q.s. Total 20 g

As the excipient, 17 g of corn starch was used.

EXAMPLE 4 Preparation of Powders

The ingredients according to the following recipe were weighed andblended to provide the composition of the invention in powdery form.

Crude soya isoflavone powder 4.1 g Galactosylsucrose (55% content) 10.0g Streptococcus E-23-17 lyophilizate 1.0 g Vitamins & minerals q.s.Flavor q.s. Total 20 g

The Streptococcus E-23-17 lyophilizate was prepared by growingStreptococcus E-23-17 (FERM BP-6436) in a suitable liquid growth medium(GAM broth) (37° C., 24-48 hours) and lyophilizing the resultingculture. The bacterial cell content of this freeze-dried powder was10⁹-10¹⁰ cells/g.

EXAMPLE 5 Preparation of Granules

The ingredients according to the following recipe were weighed andblended to provide the composition of the invention in granular form.

Crude soya isoflavone powder 4.1 g Galactosylsucrose (55% content) 10.0g Streptococcus E-23-17 lyophilizate 1.0 g Sorbitol q.s. Vitamins &minerals q.s. Flavor q.s. Total 20 g

As the Streptococcus E-23-17 lyophilizate, the same freeze-dried powderas in Example 4 was used.

EXAMPLE 6 Microbial Production of Equol

Using a water-soluble soya isoflavone material (“Fujiflavone P10”,Fujicco) as the substrate, 1 ml of a suspension of 10⁷-10⁹ cells ofStreptococcus A6G-225 (FERM BP-6437) in GAM for culture of anaerobicbacteria was added to a 2.2% aqueous solution of the above substrate.The mixture was incubated aerobically at 37° C. under stationaryconditions for 96 hours and the amount of equol produced in thefermentation broth was measured by HPLC. The concentration of daidzin inthe above aqueous solution was 1.083 mg/ml, and the concentration ofdaidzein was 0.014 mg/ml.

As a result, whereas no equol could be detected in the water-solublesoya isoflavone material, the equol content of the fermentation brothafter 96 hours of culture was 613.0±8.7 μg/ml (means of 5 determinations±S.D.). Neither daidzin nor daidzein was detected in the fermentationbroth.

Using a substrate solution containing 0.01 mg/ml of daidzein(manufactured by Funakoshi, purity ≧99%) (5 mg of daidzein suspended in2 ml of special grade methanol and diluted to 50 ml with BHI (brainheart infusion) medium) in lieu of the above water-soluble soyaisoflavone material, equol was produced in otherwise the same manner asabove. As a result, the amount of equol in the fermentation broth after96 hours of culture was 17.9±1.4 μg/ml (mean of 5 determinations ±S.D.).

It is, therefore, clear that by utilizing the microorganism of theinvention, equol can be produced from daidzein with good efficiency.

EXAMPLE 7 Preparation of a Drink

Equol-containing fermentation broth 1.55 g Glucose 5.00 g Citric acid0.5 g Vitamins & minerals q.s. Flavor q.s. Water q.s. Total 200 ml

In the same manner as Example 6, 1 ml of a suspension of 10⁷-10⁹ cellsof Streptococcus A6G-225 (FERM BP-6437) in GAM for culture of anaerobicbacteria was added to a 2.2% aqueous solution of water-soluble soyaisoflavone material (“Fujiflavone P10”, Fujicco) and the mixture wasincubated aerobically at 37° C. under stationary conditions for 96hours. Using the equol-containing fermentation broth thus obtained andother ingredient according to the above recipe, the composition of theinvention in the form of a drink was prepared.

EXAMPLE 8 Preparation of a Bar

Equol-containing fermentation broth 1.55 g Butter 20.0 g Sugar 20.0 gSalt Small amount Egg ½ Wheat flour 80.0 g Vitamins & minerals q.s.Flavor q.s. Milk 30.0 g

Using the equol-containing fermentation broth described in Example 7 inaccordance with the above recipe, a dough was prepared, molded into asuitable bar form, and baked in an oven at 170° C. for 15 minutes toprovide a cake bar.

EXAMPLE 9 Preparation of a Jelly

Equol-containing fermentation broth 1.55 g Fruit juice 50.0 g Sugar 50.0g Agar 2.5 g Vitamins & minerals q.s. Flavor q.s.

Using the equol-containing fermentation broth described in Example 7 inaccordance with the above recipe, the respective ingredients were heatedup to 90° C. with constant stirring to dissolve the agar and the wholeamount was poured into a suitable cup and cooled for gelation at 5-10°C. to provide the composition of the invention in the form of a jelly.

INDUSTRIAL APPLICABILITY

The composition of the invention, when caused to be ingested oradministered in the form of a food or a pharmaceutical product, provesuseful for the prevention and alleviation of unidentified clinicalsyndrome or menopausal syndrome in middle-aged to elderly women.

1-24. (canceled)
 25. An equol-containing composition which is obtainedby incubating at least one strain selected from the group consisting ofBacteroides E-23-15 which has been deposited as FERM BP-6435Streptococcus E-23-17 which has been deposited as FERM BP-6436 andStreptococcus A6G-225 which has been deposited as FERM BP-6437 with adaidzein-containing substance, which is in a pharmaceutical dosage form.26. The composition according to claim 25 wherein the pharmaceuticaldosage form is selected from the group consisting of aqueous solutions,emulsions, granules, powders, capsules and tablets. 27-32. (canceled)33. A method for treating menopausal syndrome in a middle-aged toelderly woman, comprising administering to a middle-aged to elderlywoman in need thereof an equol-containing composition which is obtainedby incubating at least one strain selected from the group consisting ofBacteroides E-23-15, which has been deposited as FERM BP-6435,Streptococcus E-23-17, which has been deposited as FERM BP-6436, andStreptococcus A6G-225, which has been deposited as FERM BP-6437, with adaidzein-containing substance.
 34. The method according to claim 33,wherein the equol-containing composition further contains at least onecomponent that favors the maintenance and growth of the strain.
 35. Themethod according to claim 34, wherein the component that favors themaintenance and growth of the strain is at least one substance selectedfrom the group consisting of galactosylsucrose, soybean-oligosaccharide,lactulose, lactitol and fructo-oligosaccharide.
 36. The method accordingto claim 33, wherein said daidzein-containing substance further containsat least one member selected from the group consisting of genistein,daidzin and genistin.
 37. The method according to claim 33, wherein thedaidzein-containing substance is soya isoflavone.
 38. The methodaccording to claim 33, wherein said composition is a food composition.39. The method according to claim 38, wherein the food composition isselected from the group consisting of a drink, dairy product, andfermented milk.
 40. A method for treating menopausal syndrome in amiddle-aged to elderly woman, comprising administering to a middle-agedto elderly woman in need thereof the composition of claim
 25. 41. Amethod for treating menopausal syndrome in a middle-aged to elderlywoman, comprising administering to a middle-aged to elderly woman inneed thereof the composition of claim
 26. 42. A method for treatingpostmenopausal osteoporosis in a middle-aged to elderly woman,comprising administering to a middle-aged to elderly woman in needthereof an equol-containing composition which is obtained by incubatingat least one strain selected from the group consisting of BacteroidesE-23-15, which has been deposited as FERM BP-6435, StreptococcusE-23-17, which has been deposited as FERM BP-6436, and StreptococcusA6G-225, which has been deposited as FERM BP-6437, with adaidzein-containing substance.
 43. The method according to claim 42,wherein the equol-containing composition further contains at least onecomponent that favors the maintenance and growth of the strain.
 44. Themethod according to claim 43, wherein the component that favors themaintenance and growth of the strain is at least one substance selectedfrom the group consisting of galactosylsucrose, soybean-oligosaccharide,lactulose, lactitol and fructo-oligosaccharide.
 45. The method accordingto claim 42, wherein said daidzein-containing substance further containsat least one member selected from the group consisting of genistein,daidzin and genistin.
 46. The method according to claim 42, wherein thedaidzein-containing substance is soya isoflavone.
 47. The methodaccording to claim 42, wherein said composition is a food composition.48. The method according to claim 47, wherein the food composition isselected from the group consisting of a drink, dairy product, andfermented milk.
 49. A method for treating postmenopausal osteoporosis ina middle-aged to elderly woman, comprising administering to amiddle-aged to elderly woman in need thereof the composition of claim25.
 50. A method for treating postmenopausal osteoporosis in amiddle-aged to elderly woman, comprising administering to a middle-agedto elderly woman in need thereof the composition of claim
 26. 51. Amethod for producing an equol-containing composition, comprisingincubating at least one strain selected from the group consisting ofBacteroides E-23-15, which has been deposited as FERM BP-6435,Streptococcus E-23-17, which has been deposited as FERM BP-6436, andStreptococcus A6G-225, which has been deposited as FERM BP-6437, with adaidzein-containing substance, wherein the composition is in apharmaceutical dosage form.
 52. The method according to claim 51,wherein the pharmaceutical dosage form is selected from the groupconsisting of aqueous solutions, emulsions, granules, powders, capsulesand tablets.